ES2320334T3 - USE OF FATTY ACID ANALOGS FOR THE TREATMENT AND / OR PREVENTION OF SKIN PROLIFERATIVE DISEASES. - Google Patents

Abstract

Gram-negative bacterial lipooligosaccharide detoxified and antigenic reducer with a region of detoxified lipid A and capable of being linked to an immunologically acceptable carrier by the detoxified lipid A region, said detoxified and antigenic bacterial lipooligosaccharide consisting of a portion according to the following formula: * * see formula ** in which L is the region of the detoxified lipid A Kdo is a portion of 2-keto-3-deoxyoctulosonic acid W is a portion of 2-keto-3-deoxyoctulosonic, a phosphate group or a group of pyrophosphoethanolamine Glc is glucose Hep is L-glycerol-alpha-D-hand-heptase Y is phosphoethanolamine or hydrogen X is phosphoethanolamine when Y is hydrogen and X is hydrogen when Y is phosphoethanolamine; and S 3 is N-acetyl alpha-glucosamine or L-glycerol-alpha-D-hand-heptosa.

Description

Use of fatty acid analogs for treatment and / or prevention of proliferative diseases of the skin.

Invention Sector

The present invention concerns vaccines protein-lipopolysaccharide conjugates, with a optimal presentation of oligosaccharide epitopes that show better immunogenic properties after protein coupling carriers for the lipid A region.

Background of the invention

Despite the tremendous success of protein-lipopolysaccharide conjugate vaccines in the fight against serious bacterial infections caused by encapsulated bacteria, it is not possible to apply such technology to some important bacterial pathogens, due to the fact that certain pathogenic bacteria are not encapsulated. The non-typifiable Haemophilus influenza , the main causative agent of respiratory infections and otitis media in children, belongs to this category. In addition, some bacteria have capsular polysaccharides that are poorly immunogenic even when conjugated. The α-2-8 polysalic acid capsule of group B Neisseria miningitidis, responsible for half of the cases of meningococcal meningitis in the western world, is a case in point. Gram-negative bacteria have an outer membrane that consists of components that include proteins, lipoproteins, phospholipids and glycolipids. Glycolipids consist mainly of lipopolysaccharide endotoxins (LPS). LPS are molecules composed of a) a portion of Lipid A consisting of a glucosamine disaccharide substituted with phosphate groups and long chain fatty acids in ester and amide bonds; b) a polysaccharide nucleus linked to Lipid A by means of an eight-carbon sugar, Kdo (2-keto-3-deoxyoctulosonic acid) and usually contains heptase, glucose, galactose and N-acetylglucosamine; and optionally, c) O-specific side chains composed of repeating oligosaccharide units that, depending on the genus and species of the bacterium, may contain mannose, galactose, D-glucose, N-acetylgalactosamine, N-acetylglucosamine, L-rhamnose or dideoxyhexose (abecuosa, colitosa, tivelosa, paratosa, trehalosa). In some cases, an LPS that lacks the repeating side chains -O is called a short chain lipopolysaccharide or lipooligosaccharide (LOS). In the present application the terms lipopolysaccharide (or LPS) and lipooligosaccharide (LOS) can be used interchangeably.

The main determinants are considered Gram-negative bacteria antigens reside in the complex carbohydrate structure of the LPS. These structures of Carbohydrates vary significantly, even between different species of the same genus of bacteria Gram-negative, mainly due to variations of one or more of the following factors: the composition of sugars, the oligosaccharide sequence, the link between monomer units of oligosaccharides, among them oligosaccharides, as well as substitutions / modifications of oligosaccharides

LPS is a bacterial component that offers possibilities as immunogen of a vaccine by the determinants antigens ("epitopes") found in their structures of carbohydrates However, the chemical nature of LPS prevents its use in vaccine formulations, that is, immunization active with LPS is unacceptable due to the inherent toxicity of the portion of Lipid A in some animals. The effects pathophysiological induced (directly or indirectly) by the Lipid A of the LPS in the blood flow include fever, leukopenia, leukocytosis, Schwartzman reaction, intravascular coagulation disseminated, abortion and at higher doses, shock and death.

Given the lack of capsular polysaccharide components with which to synthesize conjugate vaccines, the use of alternative carbohydrate antigens as components of conjugate vaccines is being actively investigated. One such antigen is lipopolysaccharide (LPS) or lipooligosaccharide (LOS). The LPS of N. meningitidis and H. influenzae not typed are included in the latter category (ie LOS) and both have been implicated in the immune response to natural infections caused by their respective organisms. Unfortunately, the LOS of both organisms are extremely toxic and cannot be used as vaccines, either alone or as conjugates. However, the toxicity of LOS resides solely in its portion of lipid A and strategies have been developed to overcome this problem. In the first conjugate vaccines this objective was achieved by preliminary extraction of the lipid portion A of LOS by gentle acid hydrolysis. The non-toxic truncated LOS was then conjugated to a protein thus achieved either directly or using a spacer molecule, by the terminal residue 2-keto-3-deoxyoctulosonic acid (KDO). In a more recent study, the intact LOS of non-typable H. Influenzae was detoxified, using anhydrous hydrazine before conjugation. This reagent O-deacylates the lipid portion A and thus produces the detoxification of LOS. The conjugation of the detoxified LOS was achieved by linking the carboxylic functions of an internal KDO residue to a protein by an adipic acid dihydrazide spacer. Although it has been shown that conjugates made with acid hydrolyzed LOS produce antibodies that react with their respective native LOS, some of which even show protective properties in animals, their immune response may not be optimal. This is due to the fact that the segmentation or cleavage point in the KDO residue is within the area of the internal epitopes (ie the inner core) of the LOS and therefore the structure of said epitopes can be impaired. It has been shown that internal epitopes of the inner core are very well preserved through strains of a specific species and therefore will be attractive candidates for vaccines. In addition, internal core oligosaccharides constitute a sensible choice because the structural similarity between LOS external core oligosaccharides and mammalian tissue antigens can result in poor host immune response or autoimmune diseases.

Therefore, there is a permanent need for more effective vaccines and reduced side effects. Many approaches have been adopted for a few years. One of them, described above, which has reached some popularity recently, is the partial deacilation of the lipopolysaccharide portions of the infectious microorganism. Deacilation is used to extract the esterified fatty acid side chains from lipid A, since the esterified fatty acid side chains are included among the main causes of lipid toxicity A. In US Pat. 6207157 and its publication of US Divisional Patent Application 2002 / 0001389A1 Gu et al . disclose a conjugate vaccine for nontypeable Haemophilus influenzae . It consists of a lipooligosaccharide from which the esterified fatty acid side chains of lipid A have been extracted to form a detoxified LOS (dLOS) which is then covalently linked to an immunogenic carrier. The ester bound fatty acids are extracted by hydrazine before conjugation to the adipic acid dihydrazide (ADH) binding before conjugation to an immunogenic carrier protein. In Vaccine 3463, 1-8 (2002) GU et al disclose the development of a conjugated lipooligosaccharide-based vaccine against untypifiable Haemophilus influenzae . They linked the lipooligosaccharide reacted with the adipic acid dihydrazide (AH-dLOS) to the tetanus toxoid.

Another approach has been that of Plested et al in WO01 / 22994, who disclose a vaccine for the treatment of the disease caused by Neisseria meningitidis based on elements of the internal core lipopolysaccharide. A factor that seems critical in his technique is the presence of conserved internal epitopes found in most isolates that cause disease.

Another approach has been that of Richards et al in WO02 / 16440, in which they disclose a portion of the conserved triheptosyl inner core of a lipopolysaccharide that substantially lacks the oligosaccharide chain extension of the variable outer core and its use in vaccines to prevent Haemophilus influenzae infections .

In "Infection and Immunity", March 1991, p. 843-851, Verheul et al. Disclosed a method for well-defined coupling to proteins of the phosphoethanolamine (PEA) group and the polysaccharides and oligosaccharides containing the carboxylic acid group, without the need for extensive modifications of carbohydrate antigens. The carboxylic acid group of the terminal portion 2-keto-3-deoxyoctulosonic acid was used to introduce a thiol function in lipopolysaccharide-derived oligosaccharides of the meningococcal immunotype L2 and L3, 7, 9. The oligosaccharides containing the thiol group were coupled subsequently to bromoacetylated proteins. Oligosaccharide tetanus toxoid conjugates containing the immunotype group L2 and L3, 7, 9 were made and their immunogenicities in rabbits were studied. Both conjugates of immunotype L2 and immunotype L3, 7, 9 evoked high titers of immunoglobulin G antibody (IgG) after the first booster injection. Such conjugates also showed the ability to induce long-lasting IgG antibody levels that could be detected up to 9 months after a booster injection during week 3. The Quil A adjuvant improved the immune response to all conjugates to a lesser extent, in contrast with the adjuvant effects of Quil A on these types of antigens in the mice that have been described. An elaborate conjugate of the dephosphorylated L3, 7, 9 oligosaccharides evoked a considerably lower IgG response than a similar conjugate containing PEA and enzyme-linked immunosorbent assay inhibition studies indicated a different epitope specificity. In addition, antisera elicited with whole bacteria contained antibodies directed against epitopes containing PEA, highlighting the importance of the presence of unmodified PEA groups in oligosaccharide protein conjugates derived from meningococcal lipoolisaccharides. The procedure developed offers an elegant solution for the specific coupling of oligosaccharides containing meningococcal PEA to proteins and therefore can be a very useful tool in the development of a group B meningococcal vaccine. In "Infection and Immunity", January 1984, pp. 407-412, Jennings et al. Disclosed a method by which oligosaccharides were obtained by mild acidic hydrolysis of lipopolysaccharides from a series of different strains of Neisseria meningitidis , S2, L2, L4, L5 and L10. Dephosphorylated oligosaccharides were conjugated to a tetanus toxoid as their derivatives 2-4 (4-isothiocyanophenyl) ethylamine, which resulted in the incorporation of between 18 and 38 oligosaccharides per tetanus toxoid molecule. When injected into rabbits, the conjugates produced some oligosaccharide-specific antibodies that were primarily serologically specific, but also showed some cross-reactivity. Such serological results can be attributed to regions of structural dissimilarity and similarity within oligosaccharides. The oligosaccharide-specific antibodies were also specific to the lipopolysaccharide serotype, thus indicating that oligosaccharides are the determinants associated with this specificity for serotypes. Consistent with the serological results, conjugated antisera were bactericidal for homologous serotype meningococcal organisms and in some cases for heterologous serotype organisms.

We have detected in our studies that a factor that does not seem to have been fully appreciated is the importance of coupling the carbohydrate to the carrier protein properly, so that the vital antigenic and potentially immunogenic epitopes were not modified or compromised by the strategy of conjugation used. We were specifically interested in the extraction of phosphate, pyrophosphate or pyrophosphorylethanolamine groups from the reducing end of the lipid A region of the LOS molecule, thereby creating a hemiacetal active group through which LOS can be linked to protein carriers . The fact that all known LOS and LPS contain similar terminal phosphorylated substituents in the lipid A region of the molecules means that this procedure may be important in the synthesis of conjugate vaccines, because it would have wide application possibilities. The conceptual approach is, a posteriori , surprisingly simple. Instead of using a partial epitope (for example, a released nuclear oligosaccharide) and binding it to a carrier, or using a deacylated portion (which is detoxified) but bound the carrier so that it does not maintain the integrity of the conserved inner core oligosaccharide epitope , we make sure that the deacylated (detoxified) lipopolysaccharide is bound to the protein in a place distanced from the internal epitopes by partially or completely dephosphorylating the portion of the detoxified lipid A and thus maintaining the integrity of said epitopes. It is convenient to achieve this goal with an alkaline phosphatase but any means will serve this purpose. The resulting immunogens show many hopeful possibilities. Using immunogens composed of epitopes of conserved internal core oligosaccharides presented in deacylated LPS conjugated to carrier proteins through the lipid A terminal, we have achieved considerable and surprisingly gratifying results.

Summary of the Invention

In a first aspect, the invention relates to a Gram-negative bacterial lipooligosaccharide reducer, detoxified and antigenic, with a region of lipid A detoxified likely to bind to a carrier immunologically acceptable for the lipid A region detoxified Said bacterial lipooligosaccharide Gram-negative reducer, detoxified and antigenic It consists of a portion according to the following formula:

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one

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in the that

L is the detoxified lipid A region

Kdo is a portion of acid 2-keto-3-deoxyoctulosonic

W is a portion of acid 2-keto-3-deoxyoctulosonic, a group of phosphates, or a group of pyrophosphoethanolamine

Glc is glucose

Hep is L-Glycer-? -D-Hand-Heptosa

And it is phosphoethanolamine or hydrogen

X is phosphoethanolamine when Y is hydrogen and X is hydrogen when Y is phosphoethanolamine and S3 is N-acetyl-? -Glucosamine  or L-glycerol-? -D-hand-heptosa.

The invention further relates to a vaccine conjugate to fight bacteria Gram-negative, composed of a lipopolysaccharide antigenic detoxified according to the first related aspect, bound to an immunologically acceptable carrier by the region of O-deacylated lipid A.

The invention further relates to a vaccine conjugate to fight bacteria Gram-negative, composed of a lipopolysaccharide bacterial reducing, detoxified and antigenic according to the first related aspect, linked by a carrier link immunologically acceptable for the lipid A region O-deacylated.

The invention further relates to the compounds pharmaceuticals composed of the conjugate vaccine of the invention in association with an adjuvant. The pharmaceutical compounds of the invention may further comprise an adjuvant selected from the group consisting of Freund and Ribi's adjuvant. Although the use of such adjuvants has not been approved in humans, a one skilled in the art will appreciate that it can be used in the invention other known standard adjuvants, including Aluminum compounds (ie alum), lipopolysaccharide Chemically modified, dead pertussis suspensions Bordetella, N-acetylmuramyl-L-alanyl-D-glutamine  and other adjuvants than a person of normal competition in the technique you will know.

Another embodiment of the invention is the use of a antigenic detoxified bacterial reducing lipopolysaccharide according to the invention to fight an infection Gram-negative in a mammal.

Another embodiment of the invention is the use of an antigenic detoxified bacterial reducer of the first aspect of the invention in the manufacture of a medicament for combating a Gram-negative infection in a mammal. Preferably, the mammal is a human being. The route of administration used can be intramuscular, subcutaneous, intraperitoneal, intraarterial, intravenous or intranasal; more preferably, the route of administration is intramuscular. The effective dose used may be between 10 µg and about 50 µg. The use may further comprise injection between 10 µg and about 25 µg at about 2 months and about 13 months of the administration phase. Alternatively, the use may further comprise injection between about 10 µg and about 25 µg at about 2, 4 and 16 months after the administration phase.
tion.

Although the use of hydrazine for the detoxification of LPS or LOS is described herein, the use of any reagent or enzyme capable of extracting fatty acids bound to the ester of lipid A or its attainment by genetic modification or by engineering techniques Genetics is within the scope of the present invention. The dried LPS or LOS is suspended in liquid anhydrous hydrazine at a temperature between 1 ° C and 100 ° C, preferably between 25 ° C and 75 ° C; more preferably about 37 ° C for a period of between 1 hour and 24 hours, more preferably for a period of about 2-3 hours. After the extraction of fatty acids bound to the ester, the terminal glycoside residue (for example, glucosamine) is dephosphorylated in the lipid A region of dLOS (also called LOS-OH or LPS-OH). The scope of the invention encompasses the use of any chemical reagent or chemical capable of extracting phosphate or phosphate substituents from the terminal glycoside of LOS or LPS, or by genetic modification engineering techniques. A preferred embodiment of the invention consists in using an enzyme, more preferably an enzyme from the group of alkaline phosphatase enzymes to effect the extraction of the terminal glycoside phosphate from dLOS in order to achieve the dephosphorylated analogue, P dLOS (also called LOS -OH, from P or LPS-OH, from P). The use of any linker or spacer capable of stably and efficiently conjugating dLOS to an immunogenic carrier protein is also considered. The use of linkers is known in the conjugate vaccine sector (see Dick et al , Conjugate Vaccines, JM Cruse and RE Lewis, Jr., pub. Karger, New York, pp. 48-114.

dLOS of P can be covalently linked to the carrier directly. It is possible, for example, by reductive amination as described herein. In an alternative embodiment, dLOS, of P and the carrier are separated by a link. In some cases, the presence of a spacer or link may promote improved immunogenicity of the conjugate and more efficient coupling of the dLOS of P with the carrier. The links separate the antigenic, P and carrier dLOS and, when the carrier is also an antigen, the two antigenic components, by chains whose length and flexibility can be adjusted as desired. Among bifunctional sites, the chains may contain a variety of structural features, including heteroatoms and cleavage or cleavage sites . The links also allow corresponding increases in the transfer and rotation characteristics of the antigens, increasing the access of the binding sites to soluble antibodies. Appropriate linkages include, for example, adipic acid dihydrazide (ADH), ε-aminohexanoic acid, dimethyl acetal chlorohexanol, D-glucuronolactone and p-nitrophenylamine. Coupling reagents considered for use in the present invention include hydroxy succinimides and carbodiimides. Many other linkages and coupling reagents known to persons of ordinary skill in the art are suitable for use in the invention. Dick et al . comment in detail on said compounds, ut supra .

The presence of a carrier can increase the immunogenicity of the polysaccharide and / or oligosaccharide of the LPS or LOS conjugate. In addition, raised antibodies against the carrier are medically beneficial. The polymeric immunogenic carriers may be of a natural or synthetic material containing a primary and / or secondary amino group, an azido group or a carboxyl group. The carrier can be soluble or insoluble in
Water.

You can choose from a variety of proteins immunogenic carriers for use in the conjugate vaccine of the present invention Such classes of proteins include pili, outer membrane proteins and excreted toxins from pathogenic bacteria, non-toxic forms or "toxoids" of said excreted toxins, proteins non-toxic antigenically similar to toxins bacterial (cross-reaction material or CRM) and other proteins. Non-limiting examples of bacterial toxoids considered for use in the present invention include toxin / toxoid tetanus, diphtheria toxin / toxoid, detoxified P, toxin aeruginosa A, cholera toxin / toxoid, toxin / toxoid from pertussis and the exotoxins / toxoid of Clostridium perfringens. Be they prefer the toxoid forms of said bacterial toxins. Be Consider also the use of viral proteins (i.e. hepatitis B surface / nuclear antigens; protein rotavirus VP 7) and the F and G proteins of the syncytial virus respiratory).

CRMs include CRM_197 antigenically equivalent to diphtheria toxin (Pappenheimer et al . Immunochem., 9: 891-906, 1972) and CRM 3201, a genetically engineered variant of pertussis toxin (Black et al . , Science, 240: 656-659, 1988). The scope of the invention further encompasses the use of immunogenic carrier proteins from non-mammalian sources including the hemocyanin of the Californian barnacle, the hemocyanin of the horseshoe crab and the plant edestin.

There are many coupling methods that can be considered for dLOS protein conjugates. He applicant shows in the examples set out below the selective activation of dLOS by complete extraction of the glucosamine phosphate groups of the reducing end with an alkaline phosphatase followed by reductive amination of dLOS mediated by sodium cyanoborohydride, from P to epsilon amino groups of Lysine residues exposed in TT or CRM. Alternatively, another way of making conjugates that contain a link is about dLOS cystamine derivatization, for example, by reductive amination followed by the generation of a uncle-group and conjugation of disulfide to TT N-Bromoacetylated. Other methods known in the technique for conjugating oligosaccharides that retain the integrity of the internal epitopes to the carrier proteins Immunogens also fall within the scope of the invention.

For the effective introduction of a linker, the molar ratio of linker to dLOS in the reaction mixture It is typically between 10: 1 and approximately 250: 1. Used a molar excess of the linker to ensure more coupling efficient and to limit the dLOS-dLOS coupling. In a preferred embodiment, the molar ratio is between about 50: 1 and about 150: 1; in the realization more Preferably, the molar ratio is approximately 100: 1. Be contemplate similar relationships of dLOS-TT linker in the reaction mixture. In a preferred embodiment, in the final conjugate dLOS-carrier protein, the molar ratio of dLOS, of P with the carrier is between about 15 and 75, preferably between about 25 and about 50.

The immunogenicity of the conjugates in both mice and rabbits is enhanced by the use of lipid A monophosphoryl plus trehalose dimicolate (Ribi-700; Ribi Immunochemical Research, Hamilton, Mont.) As an adjuvant. Although said adjuvant has not been approved for use in humans, an expert will appreciate that other known standard adjuvants can be used in the invention, including aluminum compounds (eg alum), chemically modified lipopolysaccharide, dead suspensions of Bordetella pertussis , N-acetylmuramyl-L-alanyl-D-glutamine and other adjuvants known to persons of ordinary skill in the art. The use of aluminum compounds is specifically preferred. Such adjuvants are described by Warren et al . (Ann. Rev. Bioche., 4: 369-388, 1986).

The dLOS carrier protein conjugates for parenteral administration may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. Said suspension may be formulated according to methods known in the art, including dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or a suspension in a parenterally acceptable diluent or solvent, such as a solution in 1.3 butanediol. Suitable diluents include, for example, water, Ringer's solution and an isotonic sodium chloride solution. In addition, sterile fixed oils can be used conventionally as a solvent or suspending medium. To this end, any soft fixed oil including synthetic mono- or diglycerides can be used. Fatty acids such as oleic acid can also be used in the preparation of injected preparations.
bles.

The conjugate vaccine according to the invention can be in soluble or microparticle form, or it can be incorporated into microspheres or microvesicles, including liposomes. Although contemplate the different routes of vaccine administration including for example, intramuscular, subcutaneous, intraperitoneal and intraarterial, the preferred form is the muscle administration In a preferred embodiment, the administered conjugate dosage will be between about 10 \ mug and about 50 \ mug of conjugated carbohydrate In a more preferred embodiment, the dose administered is between about 20 \ mug and about 40 \ mug. In the most preferred embodiment of all, the dose administered is approximately 25 \ mug. It can be administered higher doses depending on body weight. The exact dosage can be determined by the usual protocols of dosage / response known to people in a competition Normal in the art.

The vaccine according to the invention can be administer to warm-blooded mammals of any age and It is adapted to induce active immunization in mammals Young, specifically humans. As a childhood vaccine, the conjugate is administered at 2 to 4 months of age, approximately. Typically, two injections of reinforcement between about 10 \ mug and about 25 mug of conjugated carbohydrates at about 2 and then approximately 13 months from the initial injection. Alternatively, three booster injections are administered at 2, 4 and 16 months from the initial injection.

IgG antibodies elicited by the systemic administration of the conjugate vaccine will be transferred to the local mucosa and will deactivate bacterial inoculums in the mucosal surfaces (eg nasal passages). The Iga secretion will also play a role in mucosal immunity if the conjugate vaccine is given to the mucosa (i.e. intranasal form). Therefore, the conjugate vaccine will prevent local bacterial infection, as well as systemic.

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Brief description of the drawings

Figure 1 illustrates the structures of the truncated oligosaccharides (L7-OS) and length complete (L7-OH, deP) before conjugation.

Figure 2 illustrates the ELISA titles comparisons of the individual mouse sera from the immunization with either L7-OS-TT or L7-OH, from P-TT (1-10), preimmune sera (11).

Figure 3 illustrates the inhibition of L-LOS binding oligosaccharides to serum anti-L7-OH, of P-TT

Figure 4 illustrates the bacterial activity of L7-OS-TT and L7-OH antisera of P-TT against N meningitidis of strain M982B.

Figure 5a illustrates the NMR spectrum of the O-deacylated lipopolysaccharide (LPS-OH) derived from N meningitidis L3 galE without gel column filtration, showing wide unresolved lines in its NMR spectrum.

Figure 5b illustrates the NMR spectrum of the O-deacylated lipopolysaccharide (LPS-OH) derived from N meningitidis L3 galE after its gel column filtration, showing well-resolved lines consistent with the structure of the LPS-OH material.

Figure 6 illustrates the CE-MS spectrum of the dephosphorylated LPS (LPS-OH, deP) derived from N. meningitidis L3 galE.

Figure 7 shows the NMR spectra of a) LPS -OH and b) LPS-OH of P of Figure 6.

Figure 8 shows the HPLC profile of the LPS-OH, deP and CRM_ {197} over time (Tables 1 to 3) and the HPLC profile of LPS-OH, deP, joined by Kdo via a linker M2 C2H.

Figure 9 shows that the epitope of the nucleus internal was adequately represented in the conjugate as it shows the reactivity with Mab B5.

Figure 10 compares the immune response of MLC (conjugates of the lipid path A) with MLKC (conjugates of the Kdo route).

Figure 11 shows that the answer immune to epitopes of the inner core elicited by MLC It was bactericidal.

Figure 12 illustrates the passive protection of the MLC-3

Figure 13 shows an ES-MS examination of Haemophilus influenzae LPS-OH before (Fig 13a) and after (Fig. 13b) of alkaline phosphatase treatment and shows peaks indicative of dephosphorylation of LPS-OH.

Figure 14 shows the CE-MS analysis of the double-charged ions corresponding to the complete and dephosphorylated LPS-OH Haemophilus influenzae molecule, confirming that the molecule's lipid A region had lost an 80 amu species consistent with the loss of a phosphate molecule.

Figure 15 shows the 1 H-NMR spectroscopy of a sample before and after dephosphorylation indicating an efficient and specific dephosphorylation of the A?-GlcN lipid residue of Haemophilus influenzae (Fig. 15 a (phosphorylated) with Fig . 15 b (dephosphorylated).

Figure 16 shows that the LPS epitope of the inner core of Haemophilus influenzae has been adequately presented in the conjugate, as demonstrated by its reactivity with Mab LLA4.

Figure 17 shows a comparison of immune responses of SRA derived sera (conjugates of the lipid pathway A) (Fig. 17b) to SK (conjugates of the Kdo pathway) (Fig. 17a).

Figure 18 shows the immune response to Haemophilus influenza conjugates using two different adjuvants (Ribi (R) and Freunds (F)). It was clear that the immune response to said conjugates was aimed at the epitopes of the inner core in both the entire LPS molecules and the LPS -OH molecules due to the extensive cross-reactivity demonstrated by the derived sera.

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Detailed description of the invention Introduction to Examples 1 and 2

The invention of these two examples concerns meningococcal conjugated lipooligosaccharide vaccines. The importance of an adequate presentation of the internal core oligosaccharide epitopes conserved for the immunological benefits of meningococcal lipooligosaccharide-protein conjugate vaccines has been demonstrated in the following experiments. Two different oligosaccharides were obtained by chemical degradations of the same L7 lipooligosaccharide and both were terminally linked to the tetanus toxoid. One was a truncated oligosaccharide, in which the internal epitopes were incomplete and was obtained by gentle acid hydrolysis of the L7 lipooligosaccharide. This oligosaccharide was conjugated by direct reductive amination by its recently exposed terminal KDO residue. The second, a full length oligosaccharide, was obtained by O-deacylating L7 lipooligosaccharide with the subsequent extraction of phosphate substituents from its portion of lipid A using alkaline phosphatase. This allowed the full length oligosaccharide to be conjugated directly to the tetanus toxoid by reductive amination by its recently exposed terminal residue 2-N-acyl-2-deoxy-glucopyranoside (i.e. terminal glucosamine). Comparison of the immunological benefits of the two conjugates in mice showed that while both were capable of inducing significant levels of the L7 lipooligosaccharide-specific IgG antibody, only the conjugate made with the whole length saccharide could induce bactericidal antibodies against homologous meningococci. Example 2 shows that similar results were obtained using the Neisseria meningitidis strain L3 galE conjugated to CRM_197, thereby confirming the contributions of the epitopes of the internal oligosaccharides (i.e., of the inner core) to the induction of the protective antibody The LOS of the L3 galE strain of Neisseria meningitidis consists only of an internal core oligosaccharide unit that does not contain the chain extension (i.e. lacto-N-neotetraose) observed in L7. LOS Neisseria meningitidis is a human pathogen of global importance. Despite the success of the current vaccines composed of the capsular polysaccharides of groups A, C, W-135 and Y as well as the more recently developed perfected group of conjugated vaccines of C-polysaccharides (26), the group B polysaccharide is excluded of the previous vaccines although it makes an important contribution to the disease problem in developed countries (23). This fact is due to the poor immunogenicity of the group B polysaccharide in both its native form (33) and the conjugate (4, 12). Consequently, alternative vaccines based on subcapsular antigens, including lipooligosaccharides, are being investigated.

Meningococcal LOSs have been implicated in the immune response to natural infection (3, 8) but their use as vaccines is contraindicated because of their high toxicity. In addition, they exhibit considerable antigenic diversity, which constitutes another major challenge. There are currently twelve different known immunotypes (19, 32, 34, 35) among which L1-L7 are exclusively associated with group B and C meningococci and types L10-L12 are associated with group A meningococci. types L8 and L9 overlap between the two groups. The epitopes responsible for immunotyping are located in the oligosaccharide portions of LOS (13), which have been proven structurally diverse (5, 7, 14, 16, 21, 22) but also have some regions of
similarity

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Example one

Development, characterization and performance of a meningococcal vaccine based on lipopolysaccharides using strain L7 of Neisseria meningitidis conjugated to tetanus toxoid (TT) using alkaline phosphatase technology

According to the invention, in order to avoid the toxicity of LOS, the portion of the toxic lipid A was extracted by mild acid hydrolysis and subsequently the innocuous oligosaccharides were conjugated by different methods to the carrier proteins by their 2-keto-3-acid terminal residues. deoxyoctulosonic (KDO) (9, 13, 30). Although the L10 conjugates were capable of inducing antibodies of bactericidal oligosaccharides (9, 13) in mice the conjugates made with oligosaccharides associated with meningococci of groups B and C produced in comparison antisera with a sub-optimal bactericidal activity. (13-30). This fact stood out specifically in the case of L3 and L7 immunotypes, unfortunately the most common among meningococcal isolates of groups B and C (32). L3 and L7 immunotypes have similar structures, with L7 being simply the de-alkylated form of L3
(22).

A possible explanation of benefits sub-optimal immune systems of the previous conjugates is that the LSO separation point in the KDO residue is close, if not in inside, internal oligosaccharide epitopes, therefore damaging their structures. The importance of epitopes internal for the immune response has been documented (15, 24) and it is due to the structural similarity between the oligosaccharide chain  non-reducing distal of LOS and mammalian tissue antigens, what produces the immunodomain of the internal epitopes (14, 15, 20, 29, 31).

To test this hypothesis we compare the immune response of two L7 LOS-TT conjugates: one developed with a full length oligosaccharide treated with hydrazine and terminal bound (L7-OH, from P) and the other with an oligosaccharide similarly bound but truncated (L7-OS). Only the previous one could induce Bactericidal antibodies in mice.

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Materials and methods

Strains M982B (serotype L7) and 406Y (serotype L3) were grown in Bacto Todd-Hewitt broth (THB; Difco, Detroit, MI) at pH 7.3. Ten 5% chocolate agar plates (Quelab, Montreal, P.Q. (Canada) were inoculated with bacteria frozen and incubated overnight at 37 ° C in a 5% atmosphere CO 2. The bacteria were then resuspended in 50 ml of medium (THB) and transferred to an Erlenmeyer bottle with a stopper  thread containing 2 l of medium (THB). The jar was shaken for 7 h. at 37 ° C and its content was transferred to a fermenter 25 l New Brunswick Scientific MFS-128S Microferm. The bacteria were raised, killed with 1% formaldehyde and harvested by centrifugation.

LOS isolation . Some LOS of the two serotypes were isolated by a modified phenol extraction procedure, previously described (7). In the end they were purified by quadruple ultracentrifugation for 6 h. to 100,000 g. using a Beckman LE -80 ultracentrifuge.

Analytical Methods The solutions were evaporated at a reduced pressure below 40 ° C in a rotary evaporator. Gel filtration was performed on columns (1.6 x 90cm.) Of Bio-Gel P2 and P4 (Extra Fine, Bio-Rad Laboratories), using 0.02 M of a pyridine-acetate buffer (pH 5.4 ) as eluent and a flow rate of 12 ml / h. A Sephadex G-10 column (1.5 x 30 cm., Pharmacia) was used using water as an eluent and at a flow rate of 24 ml / h. Individual fractions were monitored using a Waters R403 differential refractometer.

The conjugates were analyzed with respect to its carbohydrate and protein content using the respective phenol-sulfuric acid (6) and bicinconinic acid tests. (27).

Nuclear Magnetic Resonance . NMR experiments were performed on a Bruker AMX 500 spectrometer, using a 5 mm broadband probe with the 1 H coil closest to the sample. The 1 H and 31 P NMR spectra were recorded at 300 and 340 K in 5 mm tubes at concentrations of 3-5 µg of sample in 0.5 ml D 2 O at a pH of 7.0 (1 H) and pH 7.6 (31 P). Acetone was used as the internal standard (2,225 ppm) for the 1 H spectrum. The 31 PNMR samples contained 5 mM EDTA and 2% DOC and were referenced at 85% orthophosphoric acid (0.00 ppm). All experiments were performed while the samples were rotated. The hetero-correlated 2D experiments (HSQC) were performed as previously described (2).

Chemical methods The O- deacetylation of LOS L3 and L7 was performed using anhydrous hydrazine in the manner described above (22) to achieve deactivated and partially dephosphorylated L3-OH and L7-OH O. The nuclear oligosaccharide (L7-OS) was obtained by heating the LOS (10 mg / ml) in 1% acetic acid at 100 ° C for 2 h. Insoluble lipid A was extracted by centrifugation at 15,000 rpm for 15 minutes and the water soluble L7-OS was purified on a Bio-Gel P2 column. A similar procedure was used to isolate the L3-OS from its LOS with the exception that the latter was hydrolyzed in 0.1 M acetate buffer at pH 4.2 for 2h at 100 ° C.

Enzymatic dephosphorylation of LOS-OH . LOS-OH (10 mg) were dissolved in 1 ml. 0.1 M ammonium bicarbonate (pH 8.0) and treated with 70 units of alkaline phosphatase (Boehringer Mannheim, Laval, PW Canada) at 56 ° C for 18 h. At that time 70 additional units of the same enzyme were added and the reaction was allowed to stand at 56 ° C for another six hours. The solution was then heated at 100 ° C for 5 minutes, centrifuged at 15,000 rpm for 5 minutes and the partially dephosphorylated product (LOS-OH, from P) was purified on a Sephadex G-10 column.

L7-OH coupling of P to tetanus toxoid . By the reductive amination procedures described above (12,13), L7-OH, of P was conjugated to the tetanus toxoid (TT) L7-OH, of P (10 mg) was dissolved in 200 µl of 0.02 M buffer of borate at pH 9.0, together with TT (4 mg) and sodium cyanoborohydride (50 mg). The reaction was stirred for 4 d. at 37 ° C and the progress of conjugation was monitored by HPLC using a Superosa 12 HR 10/30 column (Pharmacia) with PBS as the eluent. The conjugation was signaled by the gradual disappearance of the TT peak with the simultaneous appearance of the conjugate peak with a relatively lower K_ {av} value. Once the TT peak disappeared, the conjugate was purified on a Bio-Gel A 0.5 column (Bio-Rad) (1.6 x 42 cm.) Obtaining a yield of 5 mg. L7-OS was conjugated to TT using identical procedures
(13).

Immunization procedures Groups of 10 CF1 female mice between 6 and 8 weeks of age (Charles River, St. Constant, Canada) received a subcutaneous injection of each of the conjugates containing 2.5 µg of carbohydrates in a saline solution. Together with the complete RIBI adjuvant (RIBI Immunochem Research Inc., Hamilton, MT) in a total volume of 0.2 ml. The mice were injected on day 0, 21 and 35 and the antisera were collected on day 45, sterilized by filtration and stored at -80 ° C.

ELISA Microtiter plate wells (Lynbro / Titertek, No. 76-381-04) were coated with LOS solutions (2 µg) in a 0.05 M buffer of sodium carbohydrate at pH 9.6 at 37 ° C for 3 h and then overnight at 4 ° C. The plates were then washed and blocked with 1% BSA in a 20 mM Tris-HCl-50 mM NaCl buffer containing 0.05% Tween 20 (T_TBS) pH 7.5 for 1 h. at room temperature. The contents of the wells were then extracted and serial dilutions (100 µL / well) of murine antiserum in PBS were added and the plates were left for 2.5 hours at room temperature. After washing with T-TBS, 100 µl of a 1: 3000 dilution in PBS of an alkaline phosphatase-labeled goat IgG (H + L) (ICN, Aurora, OH) was added to each well . After incubation for 1 h. at room temperature the plates were washed with T-TBS (250 µl / well) and 100 µl / well of PNP substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added. The plates were allowed to stand for 1 h. at room temperature and the optical densities were read at 410 nm using a Dynatech MR 5000 microplate reader. Isotyping experiments were performed, using the same ELISA procedure described above with the exception of the replacement of the goat anti-isotype-specific antibodies. peroxidase-labeled mouse (Southern Biotechnology Associates Birmingham, AL.) and plate reading at 450 nm.

ELISA inhibition . The wells of the Lynbro / Titertek microtiter plates were coated with LOS, washed and blocked in the manner already described. A second microtiter plate containing duplicate serial dilutions of inhibitors in PBS (50 µl total volume) was mixed simultaneously with 50 µl of polyclonic antisera diluted 1: 100 with PBS. The plate was incubated for 1.5 h. at room temperature and the contents of the wells were transferred to the LOS coated plate already prepared. The coated plate plus the antibody and the inhibitor was incubated for 2.5 h. at room temperature and was processed and read as described in the ELISA binding experiments indicated above.

Bactericidal assay Bactericidal assays were performed in 96-well tissue culture polystyrene plates (Costar, no. 3595) essentially in the manner described above (25). N. Meningitis strain M982B was grown overnight in chocolate agar plates (Quelab, Montreal, PQ Canada) at 37 ° C in a 5% CO2 atmosphere followed by inoculation of a second plate and incubation during 5h Duplicate dilutions of murine polyclonal antisera were made directly on the plate using "Hanks' Balanced Salts" (HBSS) containing 1% casein hydrolyzate, diluted to a final volume of 50 µl / well. A suspension of group B meningococci (GBM) in HBSS, 1% casein hydrolyzate was produced, yielding an OD 490 = 0.29 and a working dilution of final bacteria was prepared by another 1: 20,000 dilution. The recently thawed bunny complement (20 µl) was added to each of the wells, followed by 30 µl of the working dilution of bacteria (2,500 CFU / well). The plate was then stirred at 37 ° C for 1 h. Subsequently, the contents of each of the wells were mixed before placing (10 µl) in the chocolate agar plates. The agar plates were incubated overnight at 37 ° C, at 5 ° C02 and the CFU number was counted. The elimination percentage was calculated in relation to the average values of either the HBSS control wells or the culture supernatant as follows: elimination percentage = (CFU_ {control} - CFU_ {mAb} / CFU_ {control}) x 100

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Results

Characterization of oligosaccharides . In the manner described above (13, 22) the application of a 1% acetic acid hydrolyzate of L7-LOS to a Bio-Gel P4 column produced an oligosaccharide (L7-OS) whose structure was confirmed by 1 H spectroscopy NMR (22). Since it contained a terminal sialic acid residue, L3-LOS was hydrolyzed under milder conditions (sodium acetate buffer at pH 4.2). Hydrolysis produced two oligosaccharides that were separated by column chromatography (Bio-Gel P4). The 1 H NMR spectra of the oligosaccharides indicated that one of them was the expected L3-OS (22) and the other its desialylated homonym (L7-OS).

The L7-LOS was also O-de- acylated using anhydrous hydrazine (22) before being further dephosphorylated by alkaline phosphatase to produce L7-OH, of P. In addition to the O- de-acylation, the structural changes made by each of the above procedures were monitored by 31 P NMR spectroscopy, in order to determine the structure of the oligosaccharide (L7-OH, of P) used for conjugation. The chemical shifts of the 31 P NMR signals of the native and modified L7-LOSs are listed in Table 1 and the assignments were made primarily by comparison with other 31 P NMR studies of both LOS (22). and lipid A (1, 11, 17, 18).

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(Table goes to page next)

2

The native L7-LOS whose structure depicted in Figure 1 showed two signals of diforosforiletanolamine at -10.36 ppm and -9.53 ppm, which were assigned to C-1 and C-4 of the portion of lipid A (17). Another signal less than -5.21 ppm was assigned to a pyrophosphomonoester group at C1 and / or C-4 of lipid A, consistent with previous assignments (17) in LOS meningococcal and with the conclusion that these positions are not all completely substituted with diphosphorylethanolamine groups (18). The signal at +0.70 ppm was assigned to a substituent of monophosphororilethanolamine at 0-3 of one of the Heptosa residues (22). The treatment of L7-LOS with hydrazine, it reduced the intensity of the signals in half C-1 and C-4 'and removed the signal from pyrophosphomonoester at -5.21 ppm (18). He also produced two new signals at +4.97 ppm and +2.53 ppm that were assigned to the esters from monophosphate to C-4 'and C-1 respectively. In this way the hydrazine treatment is capable of partially degrading defosphorylethanolamine groups to monophosphate esters.

The remaining groups of diphosphorylethanolamine in C-1 and C-4 'were eliminated completely by the first alkaline phosphatase treatment, while the monophosphorylethanolamine phosphate substituent in The heptosa residue remained intact. Some ester of monophosphate remained in C-4 ', end that was confirmed by a hetero-correlated HSQC experiment 2D, and a part had migrated to C-3 'but not There was nothing left in C-1. A second treatment with alkaline phosphatase did not remove the residual monophosphate ester in C-4 'and C-3' but the group hemiacetal terminal glucosamine residue L7-OH, of P remained completely exposed and Prepared for conjugation.

Conjugation of oligosaccharides to TT . The oligosaccharides L7-OS and L7-OH, of P, were conjugated to TT using the procedure previously described by Jennings and Lugowski (12) but with minor modifications. This time the conjugations were performed in 0.2 M borate buffer at pH 9.0, which improved the performance of the conjugates. The conjugation was easily controlled by HPLC because the peak of the conjugate was separated by 8 min. of the TT peak, and was considered complete when the TT peak disappeared. The conjugates were purified by column chromatography and their stoichiometric analysis is shown in Table 2. Both conjugates had comparable oligosaccharide / TT ratios.

TABLE 2 Conjugate Composition L7-OS-TT and L7-OH, of P-TT

3

Immunogenicity of conjugates in mice . The conjugates L7-OS-TT and L7-OH, of P-TT were evaluated for their immunogenicity in mice. Each group of mice was given three subcutaneous injections with vaccines containing 2.5 µg of carbohydrates before bleeding. The ELISA titers of the individual mice are shown in Fig. 2 using L7-LOS as a coating antigen and show that the preimmune sera contained only minute amounts of specific L7-LOS antibodies. Although both conjugates induced antibodies of the latter specificity, the levels induced by L7-OH, P-TT were consistently higher than those induced by L7-OS-TT. In addition, the subclass distribution of antibodies elicited by both conjugates was not significantly different (Table 3). Both conjugates were capable of producing high levels of potentially bactericidal antibodies IgG2a, IgG2b and IgG3.

TABLE 3 Distribution of immunoglobulin isotypes of L7-OS-TT antisera and L7-OH, from P-TT

4

Inhibition of polyclonal antisera . To characterize the murine polyclonal antisera induced by the L7-OH conjugate of P-TT, a competitive inhibition ELISA was performed using L7-LOS as the coating antigen and six different oligosaccharides obtained from L3-LOS and L7-LOS as inhibitors . The data collected from the competitive inhibition ELISA experiments using the L7-LOS oligosaccharides is provided in Figure 3. Visual inspection of the curves obtained using the L7-LOS oligosaccharides show the most important characteristic, that is, unlike L7-OH and L7-OH, of P, which were equivalent and good inhibitors of the described system, L7- OS was a lousy inhibitor.

The L3 LOS has the same structure as the L7-LOS, except that it is terminal sialylated (22). According to the inhibition results obtained using the oligosaccharides of L7-LOS, those obtained from L3-LOS exhibited inhibition properties similar within the margin of experimental error (data not shown). L3-OH and L3-OH, from P they were good inhibitors of polyclonal antiserum binding to L7-OS, while the L3-OS, as L7-OS was also a bad system inhibitor.

Bactericidal activity of antisera . The bactericidal activities of the antisera induced in mice by the conjugates L7-OS-TT and L7-OH, of P-TT against the homologous immunotype organism are shown in Figure 4. Although both conjugates could elicit specific L7-LOS antibodies that they contained potentially bactericidal isotypes, only the L7-OH-induced antiserum of P-TT was significantly bactericidal. When diluted to 1:10, it still achieved a 40% elimination, while even the undiluted antisera of the L7-OS-TT conjugate showed a very bad bactericidal activity.

Discussion

In previous studies of LOS conjugate vaccines the problem of LOS toxicity was avoided by the extraction of toxic lipid A from the molecule by mild acid hydrolysis (9, 13, 20). Conjugates were then performed using non-toxic truncated core oligosaccharides as haptens. Next, a series of different chemical procedures were used in the synthesis of the conjugates but all used the recently exposed KDO residue as a point of attachment (9, 13, 20). The conjugates were evaluated in animals and some general conclusions can be seen from the results obtained. Only conjugates made with immunotypes associated with strains of group A (L8-L12) were able to induce antibodies with good bactericidal activity (9,13). Conjugates made with immunotypes associated with strains of group B and C; like L3, 7, they induced antibodies that had no bactericidal activity (13, 30). This was disappointing because L3, 7 is the most predominant immunotype among isolates of group B disease, N, meningitidis. The ability of group A associated immunotypes to induce a bactericidal antibody is likely to be based on its structure. Unlike LOS of the strains of group B and C, which have an immunosuppressive lacto- N- neotetraose chain or its sialylated analog linked to a Hep in its interior structures (14, 16, 22), the LOS of the strains of the Group A have more immunogenic structures of the distal chain.

It is known that L3 and L7 immunotypes are closely related and only different by the addition of terminal sialic acid to the lacto-N-neotetraose chain of L3 (22). Therefore, in addition to the poor immunogenicity of the L3 and L7 immunotypes caused by the structural aspects indicated above, another factor to consider in their inability to produce bactericidal antibodies is that the cleavage of LOS in the internal KDO residue could structurally damage the epitope LOS interior. To find out this hypothesis, we conjugate an L7 oligosaccharide, after preserving internal epitopes to TT and compare the immune properties of the conjugate with those of a TT conjugate made in the manner described above with a truncated L7 nuclear oligosaccharide. To achieve our objectives we chose to combine the LOS L7 detoxified treated with hydrazine to TT. This procedure has been described above for the synthesis of non-classifiable LOS influenza conjugates of H. influenzae (10) in which they used internal KDO residues as the linkage point. However, such residues are close to the internal epitopes, if they are not part of them. Therefore, consistent with our original strategy of strictly trying to ensure the preservation of internal epitopes, we link the LOS O-de- acylated by its terminal reducing glucosamine residue. By separating the union of the region from the potential internal epitopes we hope to avoid any possibility of interference of the link with that region. In order to conjugate L7-OH to TT by reductive amination we had to extract all phosphate substituents from the C-1 hemiacetal group from the terminal glucosamine residue of lipid A. This goal was achieved by further treatment of the detoxified LOS-L7 with alkaline phosphatase. Using a combination of treatments with hydrazide and alkaline phosphatase and following the procedures by 31 P NMR spectroscopy, we were able to verify the complete extraction of both diphosphorylethanolamine groups from C-1 and C-4 'of the lipid portion A. Although two monophosphate esters remained, one located in C-4 ', which probably could have been extracted by a more appropriate treatment of enzymes, it was found that the hemiacetal in C-1 was completely free.

Therefore the previous oligosaccharide (L7-OH, from P) and L7-OS were conjugated to TT by direct reductive amination and demonstrated by analysis that both conjugates L7OH, of P-TT and L7OS-TT had some similar saccharide / TT ratios. This fact, along with their structural similarities, allowed to make a comparison Legitimate of your immune benefits. Both conjugates were capable of inducing antibodies that bound to the L7-LOS, although the conjugate that contained the epitope conserved (L7OH, of P-TT) was clearly the superior immunogen. Confirmation that the conservation of epitopes L7-LOS is a factor to obtain a effective immune response was demonstrated more dramatically at compare bactericidal activities of induced antibodies for both conjugates. The conjugate that had preserved the epitopes of internal oligosaccharide (L7-OH, of P) induced antibodies that were bactericidal to homologous organisms, while the antisera induced by the elaborated conjugate with the truncated core oligosaccharide (L7-OS-TT) were not. The latter negative result is also consistent with studies earlier using conjugates made with oligosaccharides similar of immunotypes L3 and L7 (13, 30).

The difference in the ability to induce bactericidal antibodies of the two conjugates, because both conjugates could produce antibodies that bound to L7-LOS of isotypes that had at least the possibility of being bactericidal. It may be that the lack of activity bactericide in antibodies raised by L7-OS-TT is due to its poor greed, but we have enough evidence to prove that important bactericidal epitopes are lost when the L7-OS undergoes mild acid hydrolysis.

This fact was determined in experiments of inhibition in which the different oligosaccharides were used to inhibit the binding of L7-LOS to antibodies induced by the L7-OH conjugate, of P-TT Meanwhile both the L7-OH like L7-OH, P's were equally good for the binding inhibition, L7-OS was an inhibitor Very bad of the same system. This result implies that both L7-OH as L7-OH, from P contain epitopes that are not found in L7-OS and also that these epitopes constitute the origin of a large proportion of the bactericidal antibodies When sialylated L3 oligosaccharides equivalents were replaced by those of L7 in the experiments of inhibition described above, produced curves of essentially similar inhibition, consistent with the test structural (22), that L7-LOS is equivalent to L3-LOS desialilado.

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Example 2

Development, characterization and performance of a meningococcal vaccine based on lipopolysaccharides using Neisseria meningitidis strain L3 galE conjugated to CRM 197 using alkaline phosphatase technology Materials and methods

In this example, the galE mutant of strain H44 / 76 (immunotype L3) of Neissería meningitidis was initially grown overnight at 37 ° C in 10% CO 2 on 50% Todd-Hewitt 50% Columbia agar plates (THC). Initial plates were used to ensure intensive inoculation of the initial cultures (1L) and grown in broth (THC) at 37 ° C for 18 h. The initial cultures were used to inoculate the 28L fermenter with the same media, and were grown in the same manner as the initial cultures. After its nocturnal development (17 h at 37 ° C), we killed the culture by adding phenol (1%) and cooled to 15 ° C and the bacteria were harvested by centrifugation (13, for 20 min) (Wakarchuk, W. et al ., 1996. J. Biol. Chem. 271: 19166-19173). The yields were generally 100 g biomass (wet weight). The crude LPS was extracted from the bacterial ball using the standard hot-water phenol method (Westphal, O., and JK Jann, 1965. Meth. Carbohydr. Chem. 5: 83-91) treated with Dnasa, Rnasa and Proteinase K and was purified from the aqueous phase by repeated ultracentrifuge (105,000 x G, 4 ° C, 2 x 5h) (Masoud, H., ER Moxon, A. Martin, D. Krajcarski, and JC Richards. 1997. Biochemistry 36:
2091-2103).

Preparation of LOS O-deacylated . A sample of LOS was dried overnight in vacuo and anhydrous hydrazine (98%; 1 ml / 10 mg.) LOS was added and the suspension was stirred at 37 ° C for 30-45 min., Cooled on ice and placed at cool on dry ice / acetone (acetone (5vol.) was added slowly). The solutions were centrifuged (10K, 30 min) and the ball was washed (x2) with acetone. The ball was redissolved in H2O and lyophilized to produce LOS-OH. Yield \ sim 50%.

The LPS-OH was subjected to a Quality control by ES-MS in ion mode negatives in a quadruple mass spectrometer VG Quattro (Fisons Instruments) or API 300 (Perkin-Elmer / Sciex). The samples were dissolved in 50% diluted water with acetonitril: water: methanol: 1% ammonia (4: 4: 1: 1) and the mixture was enhanced by direct infusion to 4 \ mul / min. In some cases NMR was used (although it was usually it is necessary to add EDTA and deuterated SDS to obtain a spectrum solved) in a manner similar to that described in Example 1 (previous).

Purification of O-deacylated LPS to increase the efficiency of dephosphorylation . The L3 galE LOS-PH was dissolved in H2O (~ 10 mg / 4-5 ml). The insoluble material was removed by centrifugation (14 K, 5 min). The supernatant produced was applied to a 2.5 cm Sephadex G-50 gel filtration column (medium). x 50 cm and eluted with H2O. The eluent produced was monitored by a refractive index and a PhOH / H 2 SO 4 carbohydrate assay (absorption at 490 nm). The carbohydrate positive fractions were lyophilized individually and examined by 1 H-NMR spectroscopy after dissolving them in 0.7 ml D 2 O. Fractions that offered well-resolved NMR spectra were pooled and lyophilized. Yield \ sim 50%. As Figure 5b illustrates, the NMR spectrum of the G-50 purified material was consistent with the structure of the LPS-OH material. The LPS-OH material that was not purified by column in this way showed wide unresolved lines in its NMR spectrum (Fig. 5a), indicating the possible aggregation of the material.

Dephosphorylation of alkaline phosphatase of O-deacylated LPS (LPS-OH). The methods used were basically those of Example 1 (above) with the modifications indicated below. Purified L3 O-deacylated L3 galE LPS was treated with alkaline phosphatase (Sigma; P 6772, Type VII-L of bovine intestinal mucosa, lyophilized powder purified by affinity in a tris-citrate buffer) as follows. The powder provided by the supplier was dissolved in 0.1 M of an NH 4 HCO 3 buffer (pH 8.0) to facilitate a solution of 4000 U / ml. A control reaction was always carried out by dissolving approx. 1 mg of p-nitrophenylphosphate in 1 ml of the NH 4 HCO 3 buffer (pH 8.0). 20 il of enzyme was added and in the case of the reaction, a yellow coloration was immediately observed. The purified O-deacylated LOS was dissolved in 0.1 M of an NH 4 HCO 3 buffer (pH 8.0) at a concentration of 1 mg / ml and 100 U enzyme / mg of LOS was added O-deacylated purified. The reaction mixture was stirred in a Reacti-vial at 54 ° C. After a reaction for ~ 16 h. an aliquot was extracted, heated at 100% for 5 min., cooled and centrifuged at 14 K for 5 min. to try to remove the denatured enzyme. An aliquot thereof was analyzed by CE-MS (Fig. 6) using single ion monitoring to look for diagnostic ions of the initial and dephosphorylated material. In the case of indicating a successful dephosphorylation by CE-MS, the remaining reaction mixture was denatured and centrifuged in the indicated manner and lyophilized to reduce the amount of volatile buffer present. The lyophilized material was dissolved in H2O and desalted on a Sephadex G-10 column. The eluent was monitored in the manner described above and the positive carbohydrate fractions were combined and lyophilized. The yield of the dephosphorylated products of the reaction was 80-90%. A small amount of the dephosphorylated material was examined by 1 H-NMR spectroscopy after dissolving in 0.7 ml D 2 O. An efficient dephosphorylation was verified by the observation of the loss of signal of the phosphorylated 1 H resonance corresponding to the residue of the α-GlcN lipid from 5.4 ppm (contrast Fig. 7b with Fig. 7a)

LPS-OH coupling, from P to CRM_ {197} (see Scheme 1)

Coupling by reductive amination. To a solution of CRM_197 (4.5 mg) in 0.1 N NaHCO3 (0.15 mL) was added L3 galE LOS-OH, of P (2.0 mg) and NaCNBH4 (5.0 mg). The mixture was kept at room temperature for two days. The progress of conjugation was monitored by HPLC using a Superosa 12 HR 10/30 column (Pharmacia) with PBS as eluent at a flow rate of 0.4 mL / min (Fig. 8). The conjugation was indicated by the complete disappearance of the CRM_197 peak and the simultaneous appearance of the conjugate peak with a relatively lower Kv value. The mixture was repeatedly diluted with PBS (10 ML x 4) and concentrated by Centriprep (Amicon, 50,000 Dalton) at 3000 r / min for 60 min. to extract unconjugated LPS. The final conjugate solution (2.5 mL) was subjected to column purification (Biogel A 0.5 PBS) and the fractions were collected and concentrated by Centriprep. The content of LPS and CRM197 was analyzed using the same methods described in Example 1 and the ratio of LPS to CRM197 was approximately 5.2: 1.

Scheme one

Glyco conjugation through reductive amination direct

5

The conjugation of L3 galE LOS-OH by coupling by a carboxyl of Kdo with a linker (M2C2H) (see Scheme 2) (modified procedure used by Gu et al (op. Cit.) ).

Activation of Kdo with M2 C2H. To one LPS-OH solution (2.0 mg) in water (1 mL) was added M 2 C 2 H (3 mg in 100 µL of DMSO). The pH of the solution was fixed at 4.7-4.8 using 0.01 N NaOH or 0.01N HCl EDC (3 mg in 100 µL of water) and the pH of the solution was maintained at 4.7-4.8 for 3 h. The purification was performed on a Sephadex column G-10 (40. x 1.6 cm.) Using water as eluent. Fractions containing activated LPS-OH were collected and lyophilized to amorphous material (c.a. 2.0 mg). 1 H NMR (D 2 O) showed a characteristic meileimide peak at 6.75 ppm.

CRM_ {197} thiolate. A solution of CRM_ {197} (6 mg) in 0.1 N NaHCO 3 (0.2 mL) was diluted with a buffer of triethylamine (pH 8.0) To this solution was added iminothiolane -HCL (0.5 mg) and the mixture was kept at room temperature for 2 h. The small molecules were extracted by Centriprep (10 mL x 3) until uncle compounds were detected in the wash.

Coupling. To a solution of said CRM_197 thiolate (0.8 mL) LPS-OH was added activated (2 mg) and the solution was kept at room temperature overnight. HPLC indicated a successful conjugation (see also Fig. 8). Dithioerythritol (0.5 mg) was added and subsequently performed a purification for 15 min. in a Biogel A column 0.5 Two fractions were collected and concentrated by Centriprep. The content of LPS and CRM_197 was analyzed and the ratio of LPS to CRM_ {197} was about 6.5: 1

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Scheme 2

GDOconjugation by KDO and a linker

6

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Confirmation of the presentation of the epitope . The LPS epitope of the LPS inner core was adequately presented in the conjugate as demonstrated by reactivity with MAb B5 (Fig. 9). It was previously shown that MAb B5 recognizes an epitope of the inner core conserved in meningococcal LOS in Piested et al 1999, reference No. 24 of the list of references below.

Immunization protocols They were performed according to the description of Piested et al . 1999 with the following modifications. In summary, female Balb / C mice between 6 and 8 weeks of age were immunized intraperitoneally with LPS-CRM L3 galE OdA conjugates (MLC and MLKC). Each mouse was given 2 µg of carbohydrates in 0.2 ml of Ribis complete adjuvant (Cedarlane Laboratories Ltd. Hornby, ON) by injection. The mice received a complement on day 20 and 42 of an equivalent amount of conjugate vaccine. The sera were recovered by terminal puncture of the heart on day 51.

Comparison of the immunological response to different conjugation strategies: Advantages of conjugation to the carrier protein by glucosamine coupling of the reducing end . Comparison of the immune responses of the MLC derived sera (lipid pathway conjugates) and MLKC (Kdo pathway conjugates) illustrated (Fig. 10) that the MLC derived sera generally recognize the inner core epitopes throughout the background. LPS; while MLKC derived sera do not recognize the epitopes of the inner core in the LPS background, but the immune response is directed to the O-deacylated lipid A region of the molecule, an undesirable characteristic.

Serum bactericidal assay (SB) . The bactericidal serum (SB) method was adapted from the CDC protocol with the exception that polyclonal sera derived from mice following immunizations of the conjugate were added to dilutions of human sera put in common and 1000 cfu of the Neisseria meningitidis strain with an incubation time of 40-45 min. at 37 ° C. In short, the bacteria were grown in BHI agar overnight from frozen strains. A suspension of bacteria in PBS-B was measured at OD 260 (1:50 in 1% NAOH). Using a 96 well microtiter plate, 50 µl of buffer was added to the wells in columns 2-7. 50 µl of 80% combined human decomplemented sera was added to the wells of column 1. Duplicate serial dilutions of antibody were added to columns 1-7 (discarding the last 50 µl of column 7). 1-8 50 µl of diluted bacterial suspension was added to the wells of the columns to provide 1000 cfu in 50 µl. The mixture was incubated for 40-45 minutes and placed on BHI agar plates for overnight incubation. The number of colonies on each plate was counted and the results were expressed as% cfu / ml in a decomplemented control well.

The SB trial showed that the immune response to epitopes of the inner core elicited by sera MLC -3 was bactericide. (Fig. 11).

In vivo passive protection (PP) test Passive protection model in vivo , using the five-day-old Wister infant rat model. We proceeded as described by Moe, GR et al . 1999, Infect. Immun 67: 5664-5675, except that higher doses of Neisseria meningitidis bacteria were used and a different strain of Neisseria meningitidis was used . In short, groups of five-day infant rats were randomly distributed with their mothers; weighed and administered the inoculum 1 x 10 8 cfu / ml mutant Neisseria meningitidis galE mixed 1: 1 with or (i) No antibody (PBS); (ii) irrelevant IgG3 (iii) pre-immune sera (iv and v) MLC-3 and MLC-6 polyclonal sera derived from mice after conjugate immunizations; (vi) Affinity purified B5 MAb (iv) MAb P1.2 (anti-porin antibody) (2 µg). Infant rats were monitored for signs of infection and samples were taken by bleeding from the tail vein 6 hours after infection. The animals were heavy and terminal bleeding was taken after 24 h. by cardiac puncture after an injection of pentobarbitone. The pure and diluted blood was immediately placed on BHI plates and incubated overnight. Plates were counted the next day to determine bacteremia (cfu / ml) at 6 h. and 24 h ..

The PP test shows that the answer immune to epitopes of the inner core elicited by MLC-3 serum offered passive protection against challenge for mice (Fig. 12).

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Example 3

Production, characterization and performance of a vaccine against Haemophilus influenzae based on lipopolysaccharide using strain 1003 lic1 lps A of Haemophilus influenzae conjugated to TT by alkaline phosphatase technology Introduction

Haemophilus influenzae is an important cause of the disease worldwide. Six capsular serotypes ("a" to "f") and an undetermined number of acapsular (non-typifiable) strains of H. influenzae are recognized . Type B capsular strains are associated with invasive diseases including meningitis and pneumonia, while non-typable H. influenzae (NTHi) is a leading cause of otitis media in children and respiratory tract infections in adults. Otitis media is a common childhood disease that leads to the highest frequency of visits to the pediatrician in the United States (Stool et al ., Pediatr. Infect. Dis. Suppl., 8: 211-S14, 1989).

The development of a vaccine for NTHi diseases has proved difficult due to a lack of understanding of the antigens that confer protective immunity. The effort to develop an NTHi vaccine has focused on cell surface antigens such as outer membrane proteins and pili or fimbria strains of H. influenzae (that is, against diseases caused by NTH i ) because they only protect against infections caused by H. influenzae strains that carried the type b capsule. Lipopolysaccharide (LPS) is a principal NTH i- cell surface antigen. It has been found that Haemophilus influenzae LPS contains only components of lipid A and oligosaccharide (OS). Since the LPS lipid A component is toxic, it must be detoxified before conjugation to an immunogenic carrier, as described above. In this example, a non-typable strain of H. influenzae, strain 1003 with specific mutations in the lic1 and lpsA genes is used. As disclosed by W002 / 16640, this double mutant strain of H. Influenzae produces LPS comprising the conserved region of the oligosaccharide of the inner core of H. influenzae bound only with a portion of lipid A.

Materials and methods

Unless otherwise indicated, the methods used are those described in Example 2. Strain 1003 lic1 lpsA of H.influenzae was grown at 37 ° C in a brain and heart infusion broth (BHI) with hemin supplement (10 mug / ml) and NAD (2 / / ml). For selection after transformation, kanamycin (10 µg (ml) was added to the culture medium. LOS was isolated by the phenol water extraction method, O-deacylated with anhydrous hydrazine and purified gel filtration chromatography on a Sephadex G-50 column as above
described

Enzymatic dephosphorylation of LOS-OH . The alkaline phosphatase treatment was performed essentially as described above. The ES-MS study of LPS-OH before (Fig. 13a) and after (Fig. 13b) of the alkaline phosphatase treatment showed peaks indicating that dephosphorylation of LPS -PH had taken place. Subsequent CE-MS analysis on double charged ions corresponding to the complete and dephosphorylated LPS-PH molecule confirmed that the lipid A region of the molecule had lost an 80 amu species consistent with the loss of a phosphate molecule (Fig .14).

1 H-NMR spectroscopy of the sample before and after dephosphorylation gave samples of efficient and specific dephosphorylation due to observation of the loss of the signal for the anomeric 1 H resonance phosphorylated corresponding to the reducing end α-GlcN of LOS O-deacylated to from 5.4 ppm (compare Fig. 15a (phosphorylated) with Fig. 15b (dephosphorylated)).

Coupling of Haemophilus influenzae 1003lic1 LpsA LOS-OH, de-P not typifiable to tetanus toxoid (see Scheme 3) . In this example, a spacer is used to link the LPS-OH, from -P to the TT as follows.

Activation of TT with groups of bromoacetyl (TTAcBr). To a solution of 20 mg of TT (0.5 mL) in 1.6  mL of 0.1 M Na 2 HPO 4 20 mg succinyl ester was added of acetyl bromine (Sigma) in 0.4 ml of DMSO. The mixture was stirred at room temperature for 1 h. when a TNBS test indicated a almost complete derivation of the amino groups (TNBS test: solutions of TT, TTAcBr (both at 3 mg / mL, 200 µL) and the buffer were reacted with fresh 5% TNBS in a buffer of 0.1 M borate (pH 8.3 400 µL) for 1.5 h. Only the TT showed a yellow color (420 nm)). Then the mixture was passed by a Sephadex G-25 column (1.6 x 40 cm.) using a 10 mM phosphate buffer (pH 6.5) containing 1 mM of EDTA as eluent. The first peak (protein) was collected (approximately 10 mL).

LPS derivatization with a spacer (LPS-OH, SS) . To a solution of LPS-OH, of P (10 mg) in 0.1 M NaHCO 3 (3 mL) was added cystamine (75 mg, Aldrich) and Na CNBH 3 (40 mg. Re-crystallized to guarantee its purity). The mixture was kept at room temperature for 72 h. The mixture was purified on a Sephadex G-25 column using water as eluent. The first peak was collected and lyophilized (10 mg). The CE-MS analysis indicated that the conversion yield was approximately 50-60% based on the mlz of 1084 (product) and 1016 (starting material).

Activation of LPS-PH, SS to LPS-OH, SH. The LPS-PH, SS (10 mg) derived from the spacer was dissolved in 2.0 mL of 0.1 M Na 2 HPO 4 with 0.2 M DTT at RT for 1.5 h. The solution was passed through a Sephadex G-25 column (1.6 x 40 cm.) Using 10 mM of a phosphate buffer (pH 6.5) containing 1 mM EDTA as eluent. The first peak was collected (approximately 8 mL) and the Ellman test gave a strong positive (yellow color), indicating the presence of thiol groups.

Coupling. TTAcBr solutions and LPS-OH, SH were mixed and the pH adjusted adding 0.1 M Na 2 HPO 4 up to 7.5. Mix (approximately 36 mL) was maintained at room temperature for 30 h. The HPLC profile showed a slight deviation, indicating that they had formed conjugates of a higher molecular weight.

Extraction of unconjugated LPS. The solution of indicated reaction was concentrated to about 5 mL by its centrifuged with a Centriprep (Amicon, 50,000 Dalton) at 3,000 r / min for 60 minutes. The solution was diluted by the addition of PBS (10 mL, CMF, Sigma) and was concentrated. The process was repeated. six times when carbohydrate was not detected in Last two washes. Each wash was checked by BCA test and the phenol sulfuric test. The conjugates in PBS (36 mL) were obtained with TT at 1.5 mg / mL and LPS at 0.2 mg / mL. The LPS to TT ratio was approximately 10: 1.

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Scheme 3

Glycoconjugate for lipid A and a linker

7

Coupling by Kdo and a linker. 1003 liclpsA LPS-PH of Haemophilus influenzae was also coupled to TT by the Kdo group and a spacer, using the methodology described in Example 2.

Confirmation of the presentation of epitopes . The LPS epitope of the inner core was suitably presented in the conjugate as shown by its reactivity with LLA4 MAb (Fig. 16). It has already been shown that MAb LLA4 recognizes an internal core epitope conserved in LPS of Haemophilus influenzae (Example 6 in WO02 / 16640).

Immunization protocols They essentially followed those indicated in Example 5 of WO02 / 16640. Female Balb / C mice between 6 and 8 weeks of age were immunized intraperitoneally with 1003 lic1, lpsA, OdA LPS-TT (SRA and SK) conjugates. Each mouse received 10 µg of carbohydrates in 0.2 mL of Ribi or Freunds complete adjuvant per injection. A booster was administered to the mice on day 21 and 42 with an equivalent amount of conjugate vaccine. The sera were recovered by terminal heart puncture on day 51.

Comparison of the immune response to the different conjugation strategies: Advantages of conjugation by the reducing end glucosamine . Comparison of the immune responses of SRA derived sera (conjugates of the lipid A pathway with Ribi as an adjuvant) (Fig. 17b) to SK (conjugates of the Kdo pathway) (Fig. 17a) showed that SRA derived sera usually recognize epitopes of the inner core throughout the LOS background, while the SK derived sera are directed significantly to the O-deacylated lipid A region, which does not constitute a desirable aspect. This behavior was corroborated by the observation that all SK sera recognized LOS-OH of the heterologous meningococcal L3 galE strain but did not recognize the intact LOS of this L3 galE strain, confirming that a significant proportion of the immune response induced by immunization with Conjugates 1003 lic1, lPSA OdA LPS-TT bound by the Kdo portion was directed to the O-deacylated lipid A region of the molecule.

The studies that use technology alkaline phosphatase to produce conjugates with the binding to the glucosamine of the reducing end in the lipid A region of the serving of the carbohydrate gave the following results. Using two different adjuvants (Ribi and Freunds) it became clear that the immune response to said conjugates was directed at epitopes of the inner core in both the entire LPS molecules and the LPS-OH. In Figure 18, R corresponds to Adjuvant Ribi and F with Adjuvant Frenunds and odA refers to LPS-OH molecules O-deacylated.

Cross reactivity of the derived sera.

Non-typable strains were obtained from Prof. Eskola as part of a Finnish Otitis Media Cohort Study and are mainly isolates obtained from the middle ear of children. These strains are further described in Hood et al ., Mol. Microbiol, 33.679-792, 1999. 102 strains of NTHi otitis media were sent to Richard Goldstein in Boston to be included in a study of the diversity of more than 600 capsular strains of H. influenzae and NTH i obtained worldwide and during a period of 35 years, through the analysis of ribotypes. When a dendrogram was prepared from the results of the ribotype, NTH otitis media isolates were detected in almost all branches obtained. The 25 representative strains were selected from branches that encompassed the dendrogram and therefore represent the known diversity associated with the H. influenzae species . Some selected from the same cluster were included in the 25 strains to allow an assessment of the diversity of closely related isolates.

Sera derived from immunizations with both Ribi and Freunds as adjuvants gave a broad cross-reactivity response against the LPS of the 25 non-typable strains of Haemophilus influenzae that represented the genetic diversity present in this species (Table 4).

TABLE 4

8

TABLE 4 (continuation)

9

References

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3. Brandtzaeg , PP, P. Kierulf , P. Ganstäd , A. Skulberg , JM Bruun , S. Halvorsen , and E. Sorensen . 1989 Plasma endotoxin as a predictor of multiple organ failure and death in systemic meningococcal disease. J. Infect. Dis . 160: 58-65.

4. Devi , SJ, JB Robbins , and R Schneerson 1991 . Antibodies to poly [(2-8) -? -N-acetyineuraminic acid] and poly [(2-9) -? -N-acetylneuraminic acid] are elicited by immunization of mice with Escherichia coil K92 conjugates: potential vaccines for groups B and C meningococci and E. coli K1 Natl Acad. Sci. USA 88; 7175-7179.

5. DiFabio , JL, F. Michon , J.-R. Brisson , and HJ Jennings . 1990 , Structure of the L1 and L6 oligosaccharide epitopes of Neisseria meningitidis . Dog. J. Chem . 68: 1029-1034.

6 Dubois , M., H. Gillis , JK. Hamilton , AA Rebers , and R. Smith . 1956 Colorimetricmethods for the determination of sugars and related substances. Anal. Biochem 28: 250-256.

7. Gamian , A., M. Beurret , F. Michon , J: R. Brisson , and HJ Jennings . 1992 . Structure of the L2 lipopolysaccharide core oligosaccharides of Neisseria meningitidis. J. Biol. Chem . 267: 922-925.

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11. Hoist , O., S. Moller-Loennies , B. Lindner , and H. Brade . 1993 Chemical structure of the lipid A of Escherichia coli J-5. Eur. J. Biochem . 214: 695-701.

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14. Jennings , HJ, KG Johnson , and L Kenne . 1983 The structure of an R-type oiigosacchride core obtained from some lipopolysacchride of Neisseria meningitidis . Carbohydr Res . 121: 233-241.

15. Jennings , HJ, M. Beurret , A. Gamian , and F. Michon . 1987 . Structure and immunochemistry of meningococcal hpopolysaccharides. Antonie van Leeuwenhoek 53: 519-522.

16. Kogan , G., D. Uhrin , J.-R. Brisson and HJ Jennings . 1997 Structural basis of the Neisseria meningitidis immunotypes including the L4 and L7 immunotypes. Carbahydr Res . 298: 191-199.

17. Kulshin , VA, U. Zähringer , B. Lindner , CE, Frasch , C.-M. Tsai , BA Dmitriev , and ET . Rietschel 1992 , Structural characterization of the lipid A component of pathogenic Neisseria meningitidis. J. Bacteriol 174 1793-1800.

18. L'Vov, VL, IK Verner, LY Musina, Rodinov AV, AV Ignatenko and AS Shashkcv. 1992 . Structure of the sugar-phosphate moiety of lipid A from the lipopoysaccharlde of Neisseria meningitidis group B, strain BC5S No, 125. Hydrolytic stability of phosphate and pyrophosphate substltuents. Arch, Microbiol . 157: 131-134.

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21: Michon , F., M. Beurret , A. Gamian , J.-R. Brisson and HJ Jennings . 1990 .. Structure of L5 lipopolysaccharide core ongosaccharides of Neisseria meningttidis. J. Biol. Chem . 265: 7243-7247.

22. Pavliak , V., J.-R. Brisson , D. Uhrin , and HJ Jennings . 1993 Structure of the siaiylated L3 lipopolysaccharide of Neisseria meningitidis . J. Biol. Chem . 268: 14146-14152.

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30. Verheui , AFM, AK Braaat , JM Leenhouts , P. Hoogerhout , JT Poolman , H. Snippe , and J. Verhoef . 1991 Preparation characterization, and immunogenicity of meningococcal L2 and L3,9,7 phosphoethanolamine group containing oligosaccharide protein conjugates. Infect Immun 59: 843-851.

31. Verheul , AFM, GJPH Boons , A. Van der Marel , JH Van Boom , HJ Jennings , H. Snippe , J. Verhoef , P. Hoogerhout , and JT Poolman . 1991 Minimal ollgosaccha ride structures requiredforinduction of immune responses against meningococcal immunotype L1, L2, and L3, 9 phosphoethanolamine group-containing oligosaccharide-protein conjugates. Infect Immun 59: 843-851.

32. Verheal , AFM, H. Snippe , H., and JT Poolman 1993 . Meningococcal Ilpopolysaccharide: virulence factor and potential vaccine candidate. Microbiol Revs 67: 34-49.

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Claims (21)

1. Bacterial lipooligosaccharide Gram-negative detoxified and antigenic reducer with a region of lipid A detoxified and capable of being bound to an immunologically acceptable carrier for the lipid region A detoxified, said bacterial lipooligosaccharide consisting detoxified and antigenic of a portion according to the following formula: 10 in the that L is the detoxified lipid A region Kdo is a portion of 2-keto-3-deoxyoctulosonic acid W is a portion of 2-keto-3-deoxyoctulosonic, a phosphate group or a pyrophosphoethanolamine group Glc is glucose Hep is L-Glycer-? -D-Hand-Heptosa And it is phosphoethanolamine or hydrogen X is phosphoethanolamine when Y is hydrogen and X it is hydrogen when Y is phosphoethanolamine; and S3 is N-acetyl? -Glucosamine or L-glycerol-? -D-hand-heptosa. 2. Lipooligosaccharide according to Claim 1, in which S3 is a N-acetyl-? -Glucosamine. 3. Lipooligosaccharide according to Claim 1 in which W is a portion of acid 2-keto-3-deoxyoctulosonic  and S3 is N-acetyl-? -Glucosamine. 4. Lipooligosaccharide according to Claim 1 in which W is a phosphate group. 5. Lipooligosaccharide according to Claim 1 in which X is a lateral portion of phosphoethanolamine. 6. Lipooligosaccharide according to Claim 1 in which Y is a lateral portion of phosphoethanolamine. 7. Conjugate vaccine to fight a bacterium Gram-negative comprising a lipooligosaccharide bacterial detoxified and antigenic reducer according to any one of Claims 1 to 6, linked to a carrier immunologically acceptable for the lipid A region detoxified 8. Conjugate vaccine according to Claim 7, wherein said detoxified and antigenic reducing bacterial lipooligosaccharide is a detoxified and antigenic reducing bacterial lipooligosaccharide from Neisseria meningitidis. 9. Conjugate vaccine according to Claim 7 wherein said detoxified reducing bacterial lipooligosaccharide is a detoxified and antigenic reducing bacterial lipooligosaccharide from Haemophilus influenzae. 10. Conjugate vaccine according to any of the Claims 7 to 9, wherein said immunogenic carrier is a protein. 11. Conjugate vaccine according to Claim 10, wherein said immunogenic carrier is a protein selected from the group consisting of the toxin / tetanus toxoid, a cross-reaction material (CRM), high molecular weight protein NTHi, toxin / diphtheria toxoid, detoxified P. aeruginosa toxin A, cholera toxin / toxoid, pertussis toxin / toxoid, Clostridium perfringens exotoxins / toxoid, hepatitis B surface antigen, hepatitis B nuclear antigen, VP7 rotavirus protein, F and G proteins respiratory syncytial virus. 12. Conjugate vaccine according to Claim 11, wherein said immunogenic carrier protein is the toxoid tetanic. 13. Conjugate vaccine according to Claim 11, wherein said immunogenic carrier protein is CRM 197. 14. Conjugate vaccine to fight a bacterium Gram-negative comprising a lipooligosaccharide bacterial detoxified and antigenic reducer according to any one of claims 1 to 6 linked by a linker to an immunologically acceptable carrier by the region of detoxified lipid A. 15. Conjugate vaccine according to Claim 14, wherein said linker is selected from the group consisting of M 2 C 2 H cystamine, acid di-hydrazide adipic acid, ε-aminohexanoic acid, dimethyl acetal chlorohexanol, D-glucuronolactone and p-nitrophenylethylamine. 16. Conjugate vaccine of Claim 15, in that said linker is M 2 C 2 H. 17. Conjugate vaccine according to Claim 15, in which said linker is cystamine. 18. Pharmaceutical composition composed of conjugate vaccine of any one of Claims 7 to 17 in association with an adjuvant. 19. Pharmaceutical composition according to Claim 18, wherein said adjuvant is selected from the group composed of Freund's adjuvant, alum and Ribi. 20. Pharmaceutical composition according to Claim 18 or 19, further comprising a diluent pharmaceutically acceptable. 21. Use of a bacterial lipooligosaccharide detoxified and antigenic reducer according to any of the Claims 1 to 6 in the manufacture of a medicament for fight a Gram-negative infection in a mammal.